Male DBA/1J mice were purchased from Sankyo Labo Service (Tokyo, Japan). Animals were housed in groups of 4 to 6 for periods other than those described below for behavioral tests, and maintained on a light/dark cycle of 12:12 h with food and water available ad libitum.
Detecting the circumventricular organs using fluorescein isothiocyanate
To visualize the sCVOs of DBA/1J mice, a fluorescein isothiocyanate (FITC) method was used according to a previous report . Briefly, 12-week-old mice were anesthetized using isoflurane (3% in 100% O2) and transcardially perfused with the following: first, 0.1 M phosphate-buffered saline (PBS), 5 ml; second, FITC in PBS (0.1 mg/ml), 25 ml; third, PBS, 12.5 ml; and finally, 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), 40 ml. Dissected brains were postfixed overnight and cryoprotected. Brain blocks were embedded in OCT Compound (Sakura Finetek, Tokyo, Japan) and stored at – 80 °C. Consecutive coronal sections (20-μm thick) were obtained throughout brain regions containing the third ventricle and medulla using a cryostat (CM1850; Leica Biosystems, Tokyo, Japan). Every second section was embedded in anti-fading Aqua Poly/Mount (18606; Polysciences, Warrington, PA, USA) onto coverslips.
After acclimation for 1 week, 7-week-old mice were used. In accordance with a previous report, the immunization procedure comprised two intradermal injections at the base of the tail on post-immunization day (PID) 0 and PID 21 [20, 21]. In the CIA group (n = 117), a first immunization of bovine type II collagen (200 μg/mouse; Collagen Research Center, Tokyo, Japan) dissolved in 0.1 M acetic acid (4 mg/mL) emulsified in complete Freund’s adjuvant (CFA; Becton Dickinson and Company, Franklin Lakes, NJ, USA) was administered on PID 0, with a booster immunization of bovine type II collagen dissolved in 0.1 M acetic acid emulsified in incomplete Freund’s adjuvant (IFA; Becton Dickinson and Company). In the Freund’s adjuvant group (FA group; n = 16), 0.1 M acetic acid without type II collagen emulsified in CFA (PID 0) and IFA (PID 21) were administered in the same manner. In the saline group (n = 64), an equivalent volume of saline was administered. Immunization was performed in a blinded fashion in the CIA and FA groups, but not the saline group, because of differences in appearance of the saline and emulsions. In accordance with a previous report , arthritis severity was determined using arthritis scores for all four limbs on the following scale: 0, normal; 1, swelling of digits alone or localized swelling of wrist and ankle joints; 2, swelling of both digits and wrist or ankle joints; and 3, swelling of a whole limb. “Total arthritis score” defined the sum of the scores for all four limbs. Brain analyses (described below) were performed on PID 21 and PID 35 and considered to represent the pre-onset and establishment phases, respectively, while those on PID 56 and PID 84 represented chronic phases. Several mice exhibiting ulceration around the anus caused by CFA (CIA, 6 of 117; FA, 1 of 16) were excluded from further analyses. Finally, 111 CIA mice, 15 FA mice, and 64 saline mice were used in this study.
Sucrose preference test
The sucrose preference test was performed on the basis of a previous report . Briefly, mice were moved from their group-housed cage to single-housed cages (i.e., one mouse per cage), which were equipped with two drinking bottles of identical appearance that were equally and freely accessible. Food was freely accessible and the temperature and humidity were identical to the group-housed cages. The pre-test session started at 10:00 on PID 28, and the two bottles were filled with either 2% sucrose (w/v) or plain water. The positions of the two bottles were interchanged at 19:00 every day, at which point the weight of each bottle (as an indirect measure of volume) was measured. Twelve hours later (i.e., at 07:00 the following day), the weights of bottles were measured to calculate the decrease as an estimation of the amount of liquid consumed during the dark phase. The sucrose preference of each animal was defined as the ratio of consumed sucrose-containing water to the total water volume consumed during the night of PID 33 (19:00 to 07:00 the next morning; on the fifth individual housing day) and PID 34. Bottle positions were not identical for PID 33 and PID 34. The measurement of water weight was performed in a blinded manner to the content of the water.
For immunohistochemistry, mice under anesthesia were transcardially perfused with PBS followed by 4% PFA in 0.1 M PB. After post-fixation, the brain was cryoprotected. Brain blocks were embedded in OCT compound and stored at – 80 °C. Sections containing the SFO, OVLT, or AP were obtained using a cryostat at a thickness of 20 μm. For in situ hybridization, mice under anesthesia were transcardially perfused with PBS. The unfixed medulla was dissected and frozen in isopentane on dry ice, and 16-μm coronal sections were obtained using a cryostat.
Sections were washed in PBS and then incubated in blocking solution containing 1% bovine serum albumin and 0.3% Triton X-100 in PBS for 1 h at room temperature. Subsequently, sections were incubated for 21 h at 4 °C with rabbit anti-mouse ionized calcium-binding adaptor molecule 1 (Iba-1) (1:4000; Wako Chemicals, Osaka, Japan), mouse anti-mouse glial fibrillary acidic protein (GFAP) (1:2000, G3893; Sigma–Aldrich, St. Louis, MO, USA), and/or American-hamster anti-mouse CD31 (1:100, 2HB; Developmental Studies of Hybridoma Bank, Iowa University, Iowa City, IA, USA). Rabbit nonspecific IgG (20 μg/μL, 5742S; Cell Signaling Technology, Danvers, MA, USA) was used as an isotype control for the anti-Iba-1 antibody. After rinsing in PBS, sections were incubated for 2 h at room temperature with the following secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000; Thermo Fisher Scientific, Rockford, IL, USA), Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:1000; Thermo Fisher Scientific), and/or Alexa Fluor 647-conjugated goat anti-American-hamster IgG (1:400; Jackson ImmunoResearch, West Grove, PA, USA). Sections were then washed with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml; Dojindo, Kumamoto, Japan) for nuclear staining. Slices were embedded in anti-fading Aqua Poly/Mount on coverslips.
Multiplex fluorescent in situ hybridization
Multiplex fluorescent RNAscope [Advanced Cell Diagnosis (ACD), Hayward, CA, USA; Medical & Biological Laboratories, Nagoya, Japan] was performed using probes for Iba-1 (Mm-Aif1, #319141; ACD) and IL-1β (Mm-Il1b-C2, #316891C2; ACD), in accordance with the manufacturer’s instructions. We used RNAscope probe Mm (#320881, ACD) targeting housekeeping genes, Cyclophilin B and Polr2A, for positive control and probe (#320871, ACD) for negative control. Briefly, after fixation in 10% neural-buffered formalin at 4 °C for 15 min, sections were washed with PBS, incubated in ethanol, and air-dried. Sections were incubated with protease III (diluted 1:1 with PBS) for 30 min. After additional PBS washing, probe hybridization and amplification steps were performed. Iba-1 (Alexa 488) and IL-1β (Atto 550) probe-stained sections were incubated with DAPI and mounted with Aqua Poly/Mount on coverslips.
All fluorescence images were obtained using laser-scanning confocal microscopy (FV1200; Olympus, Tokyo, Japan). Grayscale (16-bit) images were captured with a c-MOS camera (1024 × 1024 pixels, DP80; Olympus) and saved in TIFF format.
Quantitative analysis for immunohistochemistry
All image analyses were performed by a blinded examiner using ImageJ (National Institute of Mental Health, Bethesda, MD, USA). Images for Iba-1 immunosignal were captured with a 20× objective lens to identify microglia cells using the “triangle methods” with the same threshold value for all analyses. The number of microglia was counted using the “analyze particle” function in ImageJ by setting “size (pixel2)” to 75 - infinity and “circularity” to 0.0–1.00. The area of sCVOs was determined using DAPI staining in each image by identifying areas with high DAPI-positive signal density. After measuring the sCVO area, the ratios of Iba-1-immunopositive area to sCVO area (in %) and specific number of microglia (in/mm2) were calculated. Values were calculated using the mean value of 4–5 sections from each animal except for the analysis in the saline group on PID 35, where the mean value was calculated using the mean of two sections per animal.
For evaluation of microglial morphology, quantitation was performed on immunostained images using a × 40 objective lens. Regions of interest were placed on the four main divisions of the AP based upon GFAP immunostaining, as described previously (Supplementary Figure 1) [23, 24]. Binary images in each region of interest were acquired using the same threshold algorithm (at least eight regions from two slices per mouse). To extract single cell images, binary images were segmented using the “analyze particle” function by setting “size (pixel2)” as 300-infinity and “circularity” as 0.0–1.00. The following twelve morphological parameters were measured in each cell: perimeter, area, ratio of perimeter to area, ferret length, minimum ferret length, maximum and minimum diameter of approximate ellipse, aspect ratio, (minimum diameter/maximum diameter), ratio of width to height, circularity, roundness, and solidity (Supplementary Figure 2).
Quantitative analysis for multiplex fluorescent in situ hybridization
A blinded examiner performed the following image analyses. First, using the “max entropy” threshold method in ImageJ, separate binary images were created for each IL-1β and Iba-1 mRNA signals obtained after RNAscope processing. Numbers of puncta for IL-1β and Iba-1 located within DAPI-positive nuclei within the AP were counted. In accordance with the scoring guideline for RNAscope images provided by ACD, IL-1β-positive microglia cells [identified by nuclear Iba-1 and IL-1β mRNA signals (IL-1β +Iba-1+DAPI+)] were classified using the following IL-1β expression scales: “IL-1β negIba-1+DAPI+”, no-expression; “IL-1β lowIba-1+DAPI+”, coexistence of 1–3 nuclear puncta; and “IL-1β highIba-1+DAPI+”, coexistence of four or more nuclear puncta. Values from two slices from a single mouse were averaged.
Quantitative real-time polymerase chain reaction
For arthritis analysis, RNA extraction and real-time polymerase chain reaction (RT-PCR) were performed as previously described . Briefly, total RNA was extracted from four amputated limbs using a RNeasy Lipid Tissue Mini Kit (Qiagen, Tokyo, Japan). Real-time PCR was performed using an Applied Biosystem StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with Taqman probes and the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), and Actb (Mm00607939_s1).
For brain analysis, RNA extraction and RT-PCR were performed as follows. After transcardial perfusion with PBS, total RNA was extracted from the dissected medulla containing of the AP (from the rostral to caudal end) using a RNeasy Lipid Tissue Mini Kit (Qiagen). mRNA extraction from the whole brain after separating the medulla oblongata was similarly performed. RT-PCR was performed as described above with the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), TGF-β (Mm01178820_m1), Itgam (Mm00434455_m1), and Gapdh (Mm99999915_ g1).
Expression levels normalized to Actb or Gapdh were analyzed using the ΔΔCT method. mRNA expression levels were represented as values relative to the average of the saline group.
Data are expressed as mean ± SEM. All statistical analyses were performed using R (version 3.6.1; the R foundation for Statistical Computing, Vienna, Austria) and EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) . Sample sizes for the experiments on PID 21, 56, and 84 were calculated using expected effect size and variance based on data of Iba-1-immunostained area (%) in the AP on PID 35. The Kolmogorov–Smirnov test was used as a test of normality. Unpaired t-test (two-sided) was used for comparison between two groups. When the normal distribution was not confirmed, the Mann–Whitney U test was used to compare the mean ranks of two groups. Three groups were compared by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. Correlation analysis was performed using Spearman’s rank correlation. To classify cells according to morphological parameters, principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used. Frequencies of categorical variables were compared using the chi-square test. Differences were considered significant when the p value was < 0.05.